Enzyme-labeled immunoassay and device therefor

ABSTRACT

An enzyme-labeled immunoassay is performed by the steps of allowing a test sample to react with an enzyme-labeled reagent, allowing a substrate to react with the enzyme to form a signal, and immobilising the enzyme-labeled reagent, with the prevention of a further signal formation from a predetermined time on after the immobilisation of the enzyme-labeled reagent, using an enzyme inhibitor. A device for performing this enzyme-labeled immunoassay includes an absorbent material capable of transporting a developing solution by capillary action, the absorbent material including (a) a developing liquid application zone, (b) an enzyme-labeled reagent zone containing an enzyme-labeled reagent, (c) a sample receiving zone, and (d) an indicator reagent zone capable of immobilising the enzyme-labeled reagent after the reaction of the test sample with the enzyme-labeled reagent in an amount dependent on the assay result, with an enzyme inhibitor being applied to a portion in the absorbent material upstream of the enzyme-labeled reagent zone, which enzyme inhibitor prevents the formation of a signal from a predetermined time on after the enzyme-labeled reagent is immobilised at the indicator reagent zone.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to an enzyme-labeled immunoassaycomprising the steps of allowing a test sample to react with anenzyme-labeled reagent, allowing a substrate to react with the enzyme toform a signal, and immobilising the enzyme-labeled reagent, with theprevention of a further signal formation from a pre-determined time onafter the immobilisation of the enzyme-labeled reagent, using an enzymeinhibitor. The present invention also relates to a device for performingthis enzyme-labeled immunoassay.

[0003] 2. Discussion of Background

[0004] Recently immunoassays using an enzyme as a labeling material anda device comprising an absorbent material capable of transporting anenzyme-labeled reagent by capillary action, using a developing solution,are known as convenient and simple assays for the detection of analytesubstances in biological fluids, and medicinal substances in testsamples, utilizing immunoreactions (refer to, for example, JapaneseLaid-Open Patent Application 61-145459, European Patent 186,799,Japanese Laid-Open Patent Application 63-501595, U.S. Pat. No. 5,604,110and European Patent 225,054). When using a test strip comprising such adevice, for example, a test sample containing an analyte is allowed toreact with an enzyme-labeled reagent, a substrate is then allowed toreact with the enzyme to form a signal, and immobilising theenzyme-labeled reagent reacted with the test sample, using animmunoreactive substance in an indicator reagent zone, and in apredetermined period of time after the reaction of the test sample andthe enzyme-labeled reagent, the degree of the formation of the signal,such as the coloring degree of the indicator reagent zone, is measuredor evaluated, whereby the amount of the analyte contained in the testsample is assayed.

[0005] In order to prevent the formation of the signal (coloring orluminescence) except where enzyme-labeled reagent is immobilized at theindicator reagent zone, there is proposed an immunoassay test strip inwhich a signal formation inhibitor is contained therein to prevent thereaction between the enzyme and the substrate until the developingsolution reaches the indicator reagent zone (Japanese Laid-Open PatentApplication 1-503439, U.S. Pat. No. 5,641,639, European Patent 312,565,Japanese Laid-Open Application 5-149951 and European Patent 512,390). Insuch an immunoassay using the above test strip, it is possible toprevent the formation of the signal up to the indicator reagent zone byproviding a signal formation inhibitor zone upstream of the indicatorreagent zone in terms of the transport direction of the developingsolution, optionally with the provision of a signal initiation zone,whereby immunoassay can be carried out with high sensitivity for manyanalysis items.

[0006] However, in the immunoassay using the above-mentioned signalformation inhibitor, it is possible to inhibit the formation of thesignal by the reaction between the enzyme and the substrate from theinitiation of the assay with the application of the developing solutionto the strip until the developing solution reaches the indicator reagentzone, but cannot inhibit the formation of the signal which continuouslytake place in the indicator reagent zone after a specific period oftime.

[0007] Further, in the immunoassay using a coloring substrate, thedegree of the coloring of the indicator reagent zone is evaluated byvisual inspection or using a color difference meter after the reactionbetween the enzyme and the substrate for a predetermined period of time,whereby an assay result is obtained. In such a conventional immunoassay,when a negative or positive evaluation or judgement is performed, usingthe difference in the density of the coloring in the indicator reagentzone, the density of the coloring increases with time in the course ofthe assay, so that the evaluation or judgement may be changed from“negative” to “positive.” Therefore, it is necessary to perform theassay with the assay time thereof being strictly controlled when theassay is performed, evaluating the changes in the density of thecoloring in the indicator reagent zone.

[0008] Under such circumstances, there is demanded for an enzyme-labeledimmunoassay which is capable of providing an accurate assay result, withthe formation of a signal in the indicator reagent zone which dependsupon the assay result, but without further signal formation from apredetermined time on in the course of the assay.

SUMMARY OF THE INVENTION

[0009] It is therefore a first object of the present invention toprovide an enzyme-labeled immunoassay capable of providing an accurateassay result with the formation of a signal which depends upon the assayresult, without further signal formation from a predetermined time on inthe course of the assay.

[0010] A second object of the present invention is to provide a devicefor performing the above enzyme-labeled immunoassay.

[0011] The first object of the present invention can be achieved by anenzyme-labeled immunoassay comprising the steps of allowing a testsample to react with an enzyme-labeled reagent, allowing a substrate toreact with the enzyme to form a signal, and immobilising theenzyme-labeled reagent, with the prevention of a further signalformation from a predetermined time on after the immobilisation of theenzyme-labeled reagent, using an enzyme inhibitor.

[0012] The second object of the present invention can be achieved by adevice comprising an absorbent material capable of transporting adeveloping solution by capillary action, the absorbent materialcomprising (a) a developing solution application zone to which thedeveloping solution is applied, (b) an enzyme-labeled reagent zonecomprising an enzyme-labeled reagent, (c) a sample receiving zone towhich a test sample is applied, and (d) an indicator reagent zonecomprising an indicator reagent capable of immobilising theenzyme-labeled reagent after the reaction of the test sample with theenzyme-labeled reagent in an amount dependent on the assay result, whichzones are sequentially arranged in the direction of the transport of thedeveloping solution, with a substrate for an enzyme being applied to aportion in the absorbent material upstream of the enzyme-labeled reagentzone, and an enzyme inhibitor being applied to a portion in theabsorbent material upstream of the enzyme-labeled reagent zone, whichenzyme inhibitor prevents the formation of a signal which takes place bythe reaction of the enzyme and the substrate, from a predetermined timeon after the enzyme-labeled reagent is immobilised at the indicatorreagent zone.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

[0014]FIG. 1 is a cross-sectional diagram of a preferable example of atest strip for use in the enzyme-labeled immunoassay of the presentinvention.

[0015]FIG. 2 is a cross-sectional diagram of an example of a test stripused in Examples 1 and 2 of the present invention.

[0016]FIG. 3 is a graph showing the detection time of each of an FOBTpositive standard analyte (hemoglobin 20 ng/ml) and an FOBT negativestandard analyte (hemoglobin 5 ng/ml) when a test trip of the presentinvention and a comparative test strip.

[0017]FIG. 4 is a graph showing the time dependency of the degree of thecoloring of an indicator reagent zone of a test strip of the presentinvention and the time dependency of the degree of the coloring of anindicator reagent zone of a comparative test strip when an FOBT positivestandard analyte (hemoglobin 20 ng/ml) was assayed.

[0018]FIG. 5 is a graph showing the time dependency of the degree of thecoloring of an indicator reagent zone of a test strip of the presentinvention and the time dependency of the degree of the coloring of anindicator reagent zone of a comparative test strip when an FOBT negativestandard analyte (hemoglobin 5 ng/ml) was assayed.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] With reference to FIG. 1, a preferable example of a device forperforming the enzyme-labeled immunoassay of the present invention willnow be explained.

[0020] As shown in FIG. 1, the example of the device comprises a teststrip 1 comprising an absorbent material strip 2 capable of transportinga developing solution 5 by capillary action. Examples of a material forthe absorbent material strip 2 are cellulose or nitro-cellulosederivatives such as cellulose and nitro-cellulose, filter paper made ofglass fibers, and a porous film. There is no particular limitation tothe size of the absorbent material strip 2, but it is preferable thatthe absorbent material strip 2 be in the form of a strip with a width ofabout 3 to 10 mm, a length of about 30 to 100 mm and a thickness ofabout 3 μm to 1 mm for handy use thereof.

[0021] At the opposite end portions of the absorbent material strip 2,there are provided a developing solution supply zone 3 comprising adeveloping solution supply pad, and a developing solution absorbing zone10 comprising an water absorbing pad. These pads may be made of the samematerial as that for the absorbent material strip 2 or a water-absorbingmaterial such as sponge and water-absorbing unwoven cloth. Thedeveloping solution supply zone 3 and the developing solution absorbingzone 10 are usually formed thicker than the absorbent material strip 2,but it is not an essential requirement for the developing solutionsupply zone 3 and the developing solution absorbing zone 10. Thedeveloping solution supply zone 3 and the developing solution absorbingzone 10 may also be formed by forming the opposite end portions of theabsorbent material strip 2 into the developing solution supply pad andthe developing solution absorbing pad.

[0022] The enzyme inhibitor for use in the present invention can beapplied to a portion in the test strip 1 upstream of an enzyme-labeledreagent zone 8 in terms of the transport direction by capillary actionof the developing solution 5. The enzyme inhibitor can be contained ineither the developing solution 5 or the developing solution supply zone3 or both the developing solution 5 and the developing solution supplyzone 3. As the enzyme inhibitor for use in the present invention, anyenzyme inhibitor can be used as long as the inhibitor can inhibits thereaction between the enzyme of the enzyme-labeled reagent and thesubstrate. An enzyme inhibitor suitable for the enzyme-labeled reagentcan of course be selectively used.

[0023] Examples of the enzyme for use in the present invention arephosphatase such as alkaline phosphatase; peroxydase; andβ-galactosidase.

[0024] When alkaline phosphatase is used as the enzyme, there can beused as the enzyme inhibitor, phosphoric acid; phosphates such as sodiumphosphate and potassium phosphate; and phosphoric acid compounds such asphosphoric monoester, naphtholphosphoric acid, glycerophosphoric acid,phenyl phosphate, phosphoethanol amine, phosphorylcholine, and glucosephosphoric acid; and chelate compounds such asethylenediaminetetraacetic acid, phenanthroline, andethyleneglycosetetraacetic acid.

[0025] When peroxidase is used as the enzyme, reducing agents such asascorbic acid, and azide compounds such as sodium azide and potassiumazide can be used as the enzyme inhibitors.

[0026] Furthermore, when β-galactosidase is used as the enzyme, andgalactose can be used as the enzyme inhibitor.

[0027] Even when any enzyme is used, an acid or a base can be used asthe enzyme inhibitor in order to shift the pH of an indicator reagentzone 9 from the most appropriate pH for the enzyme.

[0028] When the enzyme inhibitor is applied to the developing solutionsupply zone 3, the enzyme inhibitor is applied to part of the developingsolution supply zone 3 or to its entirety and then dried to form anenzyme inhibitor zone.

[0029] When the enzyme inhibitor is applied to the developing solutionsupply zone 3, the inhibition effect of the enzyme inhibitor in theindicator reagent zone 9 does not last and is vanescent, while when theenzyme inhibitor is contained in the developing solution 5, theinhibition effect of the enzyme inhibitor lasts long. Furthermore, whenthe enzyme inhibitor is applied to the developing solution supply zone 3and is also contained in the developing solution 5, the enzyme inhibitorcan completely prevent further signal formation from a predeterminedtime on after the immobilisation of the enzyme-labeled reagent. Theposition of the application of the enzyme inhibitor can be selected asdesired in accordance with the application of the test strip 1.

[0030] The test strip 1 is further provided with a sample receiving zone7 to which a test sample 6 is applied. In the sample receiving zone 7,the absorbent material is blocked in order to prevent the non-specificadsorption of proteins, and an enzyme-labeled reagent is containedtherein in a dried state, which enzyme-labeled reagent comprises animmunoreactive substance such as an antibody or an antigen which is tobe allowed to react with an analyte such as an antigen or an antibody islabeled with an enzyme.

[0031] It is preferable that the sample receiving zone 7 also be made asan enzyme-labeled reagent zone 8 composed of an enzyme-labeled reagentpad, whereby a large quantity of the enzyme-labeled reagent can becontained therein and a large quantity of a test sample can be appliedthereto and therefore the assay sensitivity can be significantlyimproved.

[0032] There is no particular limitation to the material for theenzyme-labeled reagent pad as long as the material is water-absorbing.Examples of the material for the enzyme-labeled reagent are a sponge,water-absorbing unwoven cloth, and filter paper, made of a poroussynthesized or natural polymer compound such as polyvinyl alcohol (PVA),cellulose or nitrocellulose, or made of a composite material composed ofsuch polymer compounds. There is no particular limitation to the size ofthe enzyme-labeled reagent pad, but it is preferable that theenzyme-labeled reagent pad be of a size with a width of about 3 to 10mm, a length of about 3 to 10 mm and a thickness of about 0.5 mm to 4mm. The amount of the enzyme-labeled reagent that can be contained inthe enzyme-labeled reagent pad differs depending upon the analyte to betested, but is usually in the range of about 0.01 μg to 5 μg on a driedbasis, which is greater in amount than the case where the enzyme-labeledreagent is directly applied to the absorbent material strip 2 and dried.

[0033] The sample receiving zone 7 can be provided in the absorbentmaterial strip 2, not only in the enzyme-labeled reagent zone 8 asmentioned above, but also upstream of the water-absorbing zone 10 andupstream of the enzyme-labeled reagent zone 8, or downstream of theenzyme-labeled reagent zone 8 and upstream of the indicator reagent zone9, in terms of the transport direction of the developing solution 5.

[0034] The indicator reagent zone 9 is positioned upstream of thewater-absorbing zone 10 and downstream of the sample receiving zone 7 interms of the transport direction of the developing solution 5. Theindicator reagent zone 9 can be provided by fixing an immono-reactivesubstance, such as an antibody or an antigen which is allowed to reactwith an analyte such as an antigen or an antibody in the test sample 6,to the absorbent material strip 2 by chemical bonding or physicaladsorption. An antibody or an antigen which is allowed to react with ananalyte such as an antigen, or an antibody, may be bonded to aninsoluble carrier, for instance, by chemical bonding such as covalentbonds, or physical adsorption, and contained in the absorbent materialstrip 2 before use. Examples of such insoluble carriers are particlesprepared by insolubilizing a mixed liquid of gelatin, gum arabi andsodium hexameta-phosphate (Japanese Patent Publication 63-29223, U.S.Pat. No. 4,416,813 and European Patent 62,968), polystyrene latex,erythrocytes of various animals, and glass fiber.

[0035] As the substrate for use in the present invention, varioussubstrates can be selectively employed depending upon the enzyme to beused in combination therewith, the coloring, or the luminescencethereof.

[0036] Examples of substrates for use with alkaline phosphatase aredisodium 5-bromo-4-chloro-3-indolyl-phosphorate (BCIP), disodium5-bromo-6-chloro-3-indolyl-phosphorate, and examples of substrates foruse with peroxidase are 1,2-phenylenediamine,3,3′,5,5′-tetramethylbenzidine,2,2′-azinobis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS).

[0037] Examples of substrates for use with β-galactosidase are5-bromo-4-chloro-3-indolyl-β-galactopyranoside, and5-bromo-6-chloro-3-indolyl-β-galactopyranoside.

[0038] Such a substrate can be applied to the test strip 1, upstream ofthe enzyme-labeled reagent zone 8 in terms of the transport direction ofthe developing solution 5 in the same manner as the above-mentionedenzyme inhibitor, and can be contained in one of the developing solution5, the absorbent material strip 2, or the developing solution supply padin the developing solution supply zone 3. It is preferable to provide asubstrate zone in the absorbent material strip 2 or the developingsolution supply zone 3, to which the substrate is applied and in whichthe applied substrate is dried, in order to perform the assay with highsensitivity.

[0039] In the enzyme-labeled immunoassay using the test strip 1 of thepresent invention, the test sample 6 is applied to the sample receivingzone 7 and at the same time, the developing solution 5 is applied to thedeveloper solution supply zone 3. The developing solution 5 is held in adeveloping solution reservoir 4 which is disposed above or below thedeveloping solution supply zone 3. For simple and handy assay, thedeveloping solution reservoir 4 and the developing solution 5 areseparated by a rupturable thin film, and by rupturing the thin film withthe finger, the developing liquid 5 can be easily brought into contactwith the developing solution supply zone 3. For making this assay easierand more handy, the test strip 1 and the developing solution reservoir 4can be incorporated into a plastic case or the like in the form of akit.

[0040] As the developing solution 5 for use in the present invention,varieties of buffer solutions such as acetic acid buffer, boric acidbuffer, Tris-HCl buffer, and diethanolamine buffer, can be employed.

[0041] The test sample 6 applied to the sample receiving zone 7 reactswith the enzyme-labeled reagent in the enzyme-labeled reagent zone 8 andis carried by the developing solution 5 which flows from the developingsolution supply zone 3 toward the indicator reagent zone 9 which islocated downstream in terms of the transport direction of the developingsolution 5. The test sample 6 which has reacted with the enzyme-labeledreagent in the enzyme-labeled reagent zone 8 reaches the indicatorreagent zone 9. When the antigen or the antibody contained in the testsample 6 is trapped by an antigen or an antibody fixed to the indicatorreagent zone 9 and immobilised there, since the antigen or the antibodycontained in the test sample 6 is bonded to the enzyme-labeled reagent,the enzyme-labeled reagent is immobilized in the indicator reagent zone9. The enzyme trapped in the indicator reagent zone 9 reacts with thesubstrate contained in the developing solution 5, so that a signal suchas coloring or luminescence is formed in the indicator reagent zone 9.However, the enzyme inhibitor contained in the developing solution 5subsequently hinders the reaction between the enzyme and the substrate,so that the formation of the signal is terminated in a predeterminedperiod of time. Thus, when the antigen or the antibody to be assayed asan analyte is contained in the test sample, the formation of the signalcan be observed in the indicator reagent zone 9. The developing solution5 and other components which are not trapped by the indicator reagentzone 9 are then absorbed by the water-absorbing pad in thewater-absorbing zone 10. When no analyte such as the antigen or theantibody is contained in the test sample, the enzyme-labeled reagent isnot trapped by the indicator reagent zone 9, so that the enzyme-labeledreagent is absorbed by the water-absorbing pad in the water-absorbingzone 10 as it is and no signals are observed in the indicator reagentzone 9. By the formation of the signal or no formation of the signal,the presence or absence of the analyte such as the antigen or theantibody can be detected. The above-mentioned enzyme-labeled reagent andpart of the substrate may react with the formation of the signal beforethe enzyme-labeled reagent reaches the indicator reagent zone 9.However, the developing solution 9 in the indicator reagent zone 9contains a sufficient amount of the substrate for performing theenzyme-labeled immunoassay of the present invention.

[0042] In the above explanation, the enzyme-labeled reagent and theimmunoreactive substance immobilized in the indicator reagent zone 9 areas being the antigen or the antibody. However, the term “the antigen orthe antibody” in this specification means such substances that arecapable of performing an antigen-antibody reaction (immunoreaction) andforming an immune complex, such as polychlonal antibodies andmonochlonal antibodies, and fragments of these antibodies such as Fab,Fab and F(ab′) ₂, and hapten.

[0043] As mentioned above, analytes for the present invention aremedicinal substances, such as theophylline, phenytoin, and valproicacid; and substances in organism, for example, low-molecular weighthormones, such as thyroxine, estrogen, and estradiol, cancer markerssuch as CEA, AFP, fecal hemoglobin (for FOBT: Fecal Occult Blood Test);viruses such as human immunodeficiency virus (HIV), adult T cellleukemia virus (HTLV-1), hepatitis B virus (HBV) and hepatitis C virus(HCV); high-molecular-weight hormones, such as thyroid stimulatinghormone (TSH) and insulin, cytokine such as IL-1, IL-2 and IL-6;varieties of growth factors such as EGF and PDG; DNA and RNA of theabove-mentioned viruses; antigens of proteins relating to inflammationsuch as CRP, and antibodies corresponding to the antigens. Examples oftest samples for the assay of such antigens and antibodies are wholeblood, serum, plasma, urine, body fluids such as lymph, and fecalextract.

[0044] Other features of this invention will become apparent in thecourse of the following description of exemplary embodiments, which aregiven for illustration of the invention and are not intended to belimiting thereof.

REFERENCE EXAMPLE 1 Preparation of Rabbit Anti-hemoglobin PolychlonalAntibody (Fab)

[0045] To 2 ml of rabbit anti-hemoglobin polychlonal antibody (200 mMsodium acetate buffer (pH 4.2)) with a concentration of 1 mg/ml, 40 μgof pepsin (Boeheringer Mannheim code : 108057) was added, and themixture was incubated at 37° C. for 10 hours.

[0046] 600 μg of a fraction of F(ab) ₂ with a molecular weight of100,000 was obtained, using Superdex 200 (16/60 gel filtration column))(made by Pharmacia Co., Ltd.), which was equilibrated with a buffersolution (0.1 M sodium phosphate, 1 mM EDTA2Na buffer solution (pH 7.0))so as to adjust the pH to 7.0.

[0047] To 600 μg of the above fraction of F(ab) ₂,2-mercaptoethanolamine was added so as to adjust the concentration ofthe fraction of F(ab) ₂ to 10 mM, and the mixture was incubated at 37°C. for 2 hours. An unreacted 2-mercaptoethanolamine was removed from themixture, whereby a fraction of Fab was obtained in an amount of 500 μg.

REFERENCE EXAMPLE 2 Preparation of Maleimidized Alkaline Phosphatase

[0048] To 2.0 mg of bovine intestinum tenue alkaline phosphatase (madeby Oriental Yeast Co., Ltd.) (0.1 M sodium phosphate buffer (pH 7.0))was added 16 μl of N-succinimidyl-4-maleimide butyric acid (GMBS made byDojindo Laboratories Co., Ltd.) dissolved in DMF with a concentration of10 mg/ml, and the mixture was incubated at 25° C. for 1 hour. Anunreacted GMBS was removed from the mixture, whereby a maleimidizedalkaline phosphatase was obtained in an amount of 1480 μg.

REFERENCE EXAMPLE 3 Preparation of Alkaline Phosphatase-labeled RabbitAnti-hemoglobin Polychonal Antibody

[0049] To 500 μg of the rabbit anti-hemoglobin polychlonal antibody Fabfraction prepared in Reference Example 1, 1400 μg of the maleimidizedalkaline phosphatase prepared in Reference Example 2, and the mixturewas incubated at 25° C. for 2 hours. From this mixture, an alkalinephosphatase-labeled rabbit anti-hemoglobin polychonal antibody fractionwith an average molecular weight of 190,000 was obtained in an amount of400 μg, using Superdex 200 (16/60 gel filtration column)) (made byPharmacia Co., Ltd.).

Example 1 Immunoassay Test Strip for FOBT (Fecal Occult Blood Test

[0050] As illustrated in FIG. 2, on an absorbent material strip 12 madeof a 5 mm wide, 50 mm long cellulose film (made by Millipore Corp.), ata position of 15 mm from the right end thereof, an indicator reagentzone 19 is formed in the shape of a line so as to cross the absorbentmaterial 12 by applying thereto a rabbit anti-hemoglobin polychlonalantibody in an amount of 1.33 μg and drying the applied rabbitanti-hemoglobin polychlonal antibody. 300 ng of a solution of thealkaline phosphatase-labeled rabbit anti-hemoglobin polychonal antibodyprepared in Reference Example 3 was applied to a 5 mm wide, 5 mm longpolyvinyl alcohol (PVA) sheet and dried, whereby a pad was prepared.This pad was provided on the absorbent material strip 12 at a positionof about 25 mm from the right end thereof, which served not only as anenzyme-labeled zone 18 but also as a sample receiving zone 17 asillustrated in FIG. 2. A 10 mm wide, 20 mm long absorption pad 20 madeof a filter paper (made by Millipore Corp.) was provided on theabsorbent material strip 12 at a position of about 10 mm from the rightend thereof.

[0051] As a developing solution 15, a 0.1 MCHES(N-cyclohexyl-2-aminoethane sulfonic acid)/NaOH (pH 10.0) buffersolution was employed. The developing solution 15 was placed in adeveloping solution reservoir 14 and the developing solution reservoir14 was tightly sealed, using an aluminum sealing film, until the assaywas initiated.

[0052] A 5 mm wide, 20 mm long filter paper (made by Millipore Corp.)was attached to the absorbent material strip 12 at a position of about10 mm from the left end thereof, which constituted a developing solutionsupply zone 13 as illustrated in FIG. 2.

[0053] At the right end portion of the developing solution supply zone13, a substrate zone 13 a was formed by applying 100 μg of disodium5-bromo-4-chloro-3-indolyl phosphate (BCIP) thereto and drying theapplied BCIP as illustrated in FIG. 2, whereby an immunoassay test strip11 was prepared.

[0054] To the developing solution 15, 4 mM monosodium phthalate wasadded, and 50 mM monosodium phthalate was applied to the developingsolution supply zone 13, whereby an immunoassay test strip 11-1 of thepresent invention was prepared. The thus prepared immunoassay test strip11-1 of the present invention is represented as “Test Strip 11-1(Developing Solution+Filter Paper+)” in FIGS. 3 and 4.

[0055] The above prepared immunoassay test strip 11 was used as it wasas a comparative immunoassay test strip 11-2, that is, without addingthe above-mentioned 4 mM monosodium phthalate to the developing solution15, and without applying the above-mentioned 50 mM monosodium phthalateto the developing solution supply zone 13. The thus prepared comparativeimmunoassay test strip 11-2 is represented as “Comparative Test Strip11-2 (Developing Solution−Filter Paper−)” in FIGS. 3 and 4.

Example 2

[0056] At room temperature, 25 μl of a fecal extract containing 20 ng/mlof hemoglobin, which was used as a positive standard sample, was appliedto the sample receiving zone 17 of the immunoassay test strip 11-1 ofthe present invention, and the aluminum sealing film of the developingsolution reservoir 14 was ruptured, whereby the developing solution 15was brought into contact with the developing solution supply zone 13,and this assay was initiated.

[0057] The coloring of the indicator reagent zone 19 was visuallyinspected with time, and was also photographed, using a commerciallyavailable digital camera (Trademark “RD-175” made by Minolta Co., Ltd.)and subjected to image processing, using an image processing softwareEDAS (made by Cosmo Bio Co., Ltd.).

[0058] The results are shown in TABLE 1 and FIGS. 3 to 5.

[0059] At room temperature, 25 μl of a fecal extract containing 5 ng/mlof hemoglobin, which was used as a negative standard sample, was appliedto the sample receiving zone 17 of the immunoassay test strip 11-1 ofthe present invention, and the aluminum sealing film of the developingsolution reservoir 14 was ruptured, whereby the developing solution 15was brought into contact with the developing solution supply zone 13,and this assay was initiated.

[0060] The coloring of the indicator reagent zone 19 was visuallyinspected with time, and was also photographed, using the samecommercially available digital camera as mentioned above and subjectedto the same image processing as mentioned above.

[0061] The results are shown in TABLE 1 and FIGS. 3 to 5.

Comparative Example

[0062] At room temperature, 25 μl of a fecal extract containing 20 ng/mlof hemoglobin, which was the same positive standard sample as used inExample 2, was applied to the sample receiving zone 17 of thecomparative immunoassay test strip 11-2, and the aluminum sealing filmof the developing solution reservoir 14 was ruptured, whereby thedeveloping solution 15 was brought into contact with the developingsolution supply zone 13 and this assay was initiated.

[0063] The coloring of the indicator reagent zone 19 was visuallyinspected with time, and was also photographed, using the same digitalcamera as used in Example 2 and subjected to the same image processingas in Example 2, using the same image processing software EDAS (made byCosmo Bio Co., Ltd.).

[0064] The results are shown in TABLE 1 and FIGS. 3 to 5.

[0065] At room temperature, 25 μl of a fecal extract containing 5 ng/mlof hemoglobin, which was the same negative standard sample as used inExample 2, was applied to the sample receiving zone 17 of thecomparative immunoassay test strip 11-2, and the aluminum sealing filmof the developing solution reservoir 14 was ruptured, whereby thedeveloping solution 15 was brought into contact with the developingsolution supply zone 13, and this assay was initiated.

[0066] The coloring of the indicator reagent zone 19 was visuallyinspected with time and was also photographed, using the samecommercially available digital camera as mentioned above, and subjectedto the same image processing as mentioned above.

[0067] The results are shown in TABLE 1 and FIGS. 3 to 5. TABLE 1Evaluation of Results (Assay time: 8 minutes) Comparative Test TestStrip 11-1 Strip 11-2 (Developing (Developing Solution + Solution −Filter Paper +) Filter Paper −) Negative Standard − + (concentration ofhemoglobin: 5 ng/ml) Positive Standard + + (concentration of hemoglobin:20 ng/ml)

[0068] As can be seen from the above, when Test Strip 11-1 (DevelopingSolution+Filter Paper+) was used, a clear cut distinction was madebetween the negative standard and the positive standard in the course ofthe 8-minute assay time, while when Test Strip 11-2 (DevelopingSolution−Filter Paper−) was used, no distinction was made between thenegative standard and the positive standard in the course of the8-minute assay time.

[0069]FIG. 3 shows that when Test Strip 11-1 (Developing Solution+FilterPaper+) was used, the difference in the detection time between thepositive standard and the negative standard was 440 seconds, and withrespect to the negative standard, the evaluation result remainednegative for 15 minutes after the imitation of the assay, while whenTest Strip 11-2 (Developing Solution−Filter Paper−) was used, thedifference in the detection time between the positive standard and thenegative standard was only 41 seconds.

[0070]FIGS. 4 and 5 show that when Test Strip 11-1 (DevelopingSolution+Filter Paper+) was used, the coloring did not develop after apredetermined time on from the detection time, with respect to both thepositive standard and the negative standard, while when Test Strip 11-2(Developing Solution−Filter Paper−) was used, the coloring developedwith time.

[0071] Thus, in the enzyme-labeled immunoassay using the test strip ofthe present invention, the change in the coloring of the indicatorreagent zone is much smaller than that in the conventionalenzyme-labeled immunoassay after a predetermined time on from thedetection time, so that the evaluation or judgement can be performedeven after the detection time. For example, in the case of FOBT (FecalOccult Blood Test), the coloring of the indicator reagent zone can besubstantially stopped after a predetermined negative or positivejudgement (for instance 8 minutes) on, so that the judgement can beperformed anytime after the period of 8 minutes. Thus, theenzyme-labeled immunoassay of the present invention is effective forperforming simple and accurate positive or negative judgement orevaluation of an analyte in accordance with the difference in the colordeveloped in the indicator reagent zone, without the necessity for thecontrol of the detection time.

[0072] Japanese Patent Application No. 9-118646 filed Apr. 23, 1997 ishereby incorporated by reference.

What is claimed is:
 1. An enzyme-labeled immunoassay comprising thesteps of: allowing a test sample to react with an enzyme-labeledreagent, allowing a substrate to react with said enzyme to form asignal, and immobilising said enzyme-labeled reagent, with theprevention of a further signal formation from a predetermined time onafter the immobilisation of said enzyme-labeled reagent, using an enzymeinhibitor.
 2. The enzyme-labeled immunoassay as claimed in claim 1 ,wherein said enzyme is phosphatase, and said enzyme inhibitor isselected from the group consisting of a phosphoric acid compound and achelate compound.
 3. The enzyme-labeled immunoassay as claimed in claim2 , wherein said phosphoric acid compound is selected from the groupconsisting of phosphoric acid, phosphate, phosphoric monoester,naphtholphosphoric acid, glycerophosphoric acid, phenylphosphate,phosphoethanol-amine, phosphorylcholin, and glycose phosphoric acid. 4.The enzyme-labeled immunoassay as claimed in claim 2 , wherein saidchelate compound is selected from the group consisting ofethylenediaminetetraacetic acid, phenanthroline, andethyleneglycosetetraacetic acid.
 5. The enzyme-labeled immunoassay asclaimed in claim 1 , wherein said enzyme is peroxidase, and said enzymeinhibitor is selected from the group consisting of a reducing compoundand an azide compound.
 6. The enzyme-labeled immunoassay as claimed inclaim 1 , wherein said enzyme is β-galactosidase, and said enzymeinhibitor is galactose.
 7. A device for performing an enzyme-labeledimmunoassay comprising an absorbent material capable of transporting adeveloping solution by capillary action, said absorbent materialcomprising (a) a developing liquid application zone to which saiddeveloping liquid is applied, (b) an enzyme-labeled reagent zonecomprising an enzyme-labeled reagent, (c) a sample receiving zone towhich a test sample is applied, and (d) an indicator reagent zonecomprising an indicator reagent capable of immobilising saidenzyme-labeled reagent after the reaction of said test sample with saidenzyme-labeled reagent in an amount dependent on the assay result, whichzones are sequentially arranged in the direction of the transport ofsaid developing solution, with a substrate for an enzyme being appliedto a portion in said absorbent material upstream of said enzyme-labeledreagent zone, and an enzyme inhibitor being applied to a portion in saidabsorbent material upstream of said enzyme-labeled reagent zone, whichenzyme inhibitor prevents a signal formation which occurs by thereaction of said enzyme and said substrate, from a predetermined time onafter said enzyme-labeled reagent is immobilised at said indicatorreagent zone.
 8. The device as claimed in claim 7 , wherein saiddeveloping solution further comprises said enzyme inhibitor.
 9. Thedevice as claimed in claim 7 , further comprising an enzyme inhibitorzone comprising said enzyme inhibitor, which is disposed upstream ofsaid enzyme-labeled reagent zone.
 10. The device as claimed in claim 7 ,wherein said enzyme is phosphatase, and said enzyme inhibitor isselected from the group consisting of a phosphoric acid compound and achelate compound.
 11. The device as claimed in claim 10 , wherein saidphosphoric acid compound is selected from the group consisting ofphosphoric acid, phosphate, phosphoric monoester, naphtholphosphoricacid, glycerophosphoric acid, phenylphosphate, phosphoethanol-amine,phosphorylcholin, and glycose phosphoric acid.
 12. The device as claimedin claim 10 , wherein said chelate compound is selected from the groupconsisting of ethylenediaminetetraacetic acid, phenanthroline, andethyleneglycosetetraacetic acid.
 13. The device as claimed in claim 7 ,wherein said enzyme is peroxidase, and said enzyme inhibitor is selectedfrom the group consisting of a reducing compound and an azide compound.14. The device as claimed in claim 7 , wherein said enzyme isβ-galactosidase, and said enzyme inhibitor is galactose.